Angiogenesis is a fundamental process required for normal growth and development of tissues, and involves the proliferation of new capillaries from pre-existing blood vessels. Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis, and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.
On the other hand, promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor α (TGF α), and hepatocyte growth factor (HGF). See, for example, Folkman et al., “Angiogenesis”, J. Biol. Chem., 267: 10931-10934, 1992, for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF family, and appear to act via endothelial receptor tyrosine kinases (RTKs). At least four vascular endothelial growth factor subtypes have been identified.
Vascular endothelial growth factor (VEGF), now known as VEGF-A, has been isolated from several sources. VEGF-A shows highly specific mitogenic activity on endothelial cells, and can stimulate the whole sequence of events leading to angiogenesis. In addition, it has strong chemoattractant activity towards monocytes, can induce plasminogen activator and plasminogen activator inhibitor in endothelial cells, and can also influence microvascular permeability. Because of the latter activity, it is also sometimes referred to as vascular permeability factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara et al., “The Vascular Endothelial Growth Factor Family of Polypeptides”, J. Cell. Biochem., 47: 211-218, 1991, and Connolly, “Vascular Permeability Factor: A Unique Regulator of Blood Vessel Function”, J. Cellular Biochem., 47: 219-223, 1991.
More recently, three further members of the VEGF family have been identified. These are designated VEGF-B, described in International Patent Application No. PCT/US96/02957 (WO 96/26736) by Ludwig Institute for Cancer Research and The University of Helsinki, VEGF-C, described in Joukov et al., EMBO J., 1996 15: 290-298, and VEGF2, described in International Patent Application No. PCT/US94/05291 (WO 95/24473) by Human Genome Sciences, Inc. VEGF-B has closely similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF. In particular, VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences.
VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique, screening for cellular proteins which might interact with cellular retinoic acid-binding protein type I (CRABP-I). Its isolation and characteristics are described in detail in PCT/US96/02597 and in Olofsson et al., Proc. Natl. Acad. Sci., 1996 93: 2576-2581.
VEGF-C was isolated from conditioned media of PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase Flt-4, using cells transfected to express Flt-4. VEGF-C was purified using affinity chromatography with recombinant Flt-4, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 15: 290-298, 1996.
VEGF-C is synthesized as a preproprotein in which the receptor binding VEGF homology domain (VHD) is flanked by amino- and carboxyl-terminal propeptides. Biosynthesis of the mature VHD involves proteolytic removal of the propeptides and results in greatly increased affinity of the VHD for VEGFR-2 and VEGFR-3 relative to the unprocessed, full length form (Joukov et al., (1997) EMBO 16: 3898-3911). Therefore, proteolytic processing activates VEGF-C. It has been suggested that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 1996 15: 290-298).
VEGF2 was isolated from a highly tumorgenic, estrogen-independent human breast cancer cell line. While this molecule is stated to have about 22% homology to PDGF and 30% homology to VEGF, the method of isolation of the gene encoding VEGF2 was unclear, and no characterization of the biological activity was disclosed.
Vascular endothelial growth factors appear to act by binding to receptor tyrosine kinases of the PDGF-receptor family. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely Flt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt-4 (VEGFR-3), Tie, and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity necessary for signal transduction.
The specific role in vasculogenesis and angiogenesis of Flt-1, Flk-1, Tie, and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and placenta growth factor (P1GF). VEGF-C has been shown to be the ligand for Flt-4 (VEGFR-3), and also activates VEGFR-2 (Joukov i., 15: 290-298, 1996). A ligand for Tek/Tie-2 has been described (International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc.); however, the ligand for Tie has not yet been identified.
The receptor Flt-4 is expressed in venous and lymphatic endothelia in the fetus, and predominantly in lymphatic endothelia in the adult (Kaipainen et al., Cancer Res., 1994 54: 6571-6577; Proc. Nat. Acad. Sci. USA, 92: 3566-3570, 1995).
Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that binds and activates VEGF receptor-2 (VEGFR-2) and VEGFR-3 (Achen et al., Proc. Natl. Acad. Sci. USA 95: 548-553, 1998), cell surface receptor tyrosine kinases expressed predominantly on blood vascular and lymphatic endothelia respectively (for review see Stacker et al., FASEB J. 16: 922-934, 2002). VEGFR-3 signals for lymphangiogenesis (growth of lymphatic vessels) (Veikkola et al., EMBO J. 20: 1223-1231, 2001) whereas VEGFR-2 is thought to signal for angiogenesis (growth of blood vessels). As would be expected given the receptor specificity of human VEGF-D, this growth factor stimulates both angiogenesis and lymphangiogenesis (1Byzova et al, Blood 99: 4434-4442, 2002; Veikkola et al, EMBO J. 20: 1223-1231, 2001; Marconcini et al., Proc. Nat. Acad. Sci. USA 96: 9671-9676, 1999)
Importantly, VEGF-D stimulated tumor angiogenesis that enhanced solid tumor growth and induced lymphangiogenesis that promoted metastatic spread of tumor cells to the lymphatics and lymph nodes (Stacker et al., Nature Med. 7: 186-191, 2001). Recently, VEGF-D expression was reported to be an independent prognostic factor for both overall and disease-free survival in colorectal cancer (White et al., Cancer Res. 62: 1669-1675, 2002).
VEGF-D is secreted from the cell in a relatively inactive form containing an N-terminal propeptide, a C-terminal propeptide, and a central VEGF homology domain (“VHD”) containing the binding sites for VEGFR-2 and VEGFR-3 (Achen, M. G., M. Jeltsch, E. Kukk, T. Mäkinen, A. Vitali, A. F. Wilks, K. Alitalo, and S. A. Stacker. 1998. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk-1) and VEGF receptor 3 (Flt-4). Proc. Natl. Acad. Sci. USA 95:548-553, Joukov, V., T. Sorsa, V. Kumar, M. Jeltsch, L. Claesson-Welsh, Y. Cao, O, Saksela, N. Kalkkinen, and K. Alitalo. 1997. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16:3898-3911, and Stacker et al., J. Biol. Chem. 274: 32127-32136, 1999.) Subsequently, the propeptides are proteolytically cleaved from the VHD to generate a mature form, consisting of dimers of the VHD, that binds VEGFR-2 and VEGFR-3 with high affinity. The affinities of the mature form for VEGFR-2 and VEGFR-3 are approximately 290-fold and 40-fold greater, respectively, than those of the unprocessed form (Stacker et al., 1999, supra).
Therefore, proteolytic processing of both VEGF-D and VEGF-C is a mechanism for activating the growth factors. The proteases involved in this activation process, however, were unknown. This activation may also be involved in various biological processes, including modulating protein localization, bioavailability, and receptor specificity. These processes, in turn, may be associated with various diseases. Selective inhibition and/or activation of these processes and activators thereof will provide treatment options for patients in need thereof.
A provisional matrix is known to play a key role in angiogenesis. The provisional matrix serves as substrate for adhesion, migration and invasion of endothelial cells, and is also essential for endothelial cell survival. The provisional matrix is continuously generated and broken down, a process known as remodeling, until a new vessel is properly formed.
Remodeling of the provisional matrix is highly regulated through the balanced action of numerous molecules. Plasmin, a serine protease formed through activation of its zymogen plasminogen, plays a key role by mediating degradation of the provisional matrix. Because of its critical role in the remodeling process of the provisional matrix, plasmin level is tightly controlled through an intricate coordination between plasminogen activators, plasminogen activator inhibitors and plasmin inhibitors, see e.g. International Patent Application WO 01/62799 A2.